In Vitro And In Vivo Evaluation Of Cellular Uptake And Transport Mechanism Of Nano-delivery Systems Across The Round Window Memabrne For Inner Ear Delivery

In recent years,the application of nanosystems in the treatment of inner ear diseases has developed rapidly.As a biomembrane barrier for substances to enter the middle ear and inner ear,the round window membrane(RWM)makes it difficult for most drugs to penetrate into the RWM and enter the inner ear,thus reducing the therapeutic effect.Therefore,it is very important to study the fate of nanosystem entering the RWM to improve transport efficiency and to treat inner ear diseases.However,the current understanding of the process of transporting nanosystems from the middle ear to the inner ear,as well as the transport behavior in vitro and in vivo,is still lacking.Understanding the interaction mechanism between nanosystems and RWM still poses great challenges.In this paper,through the study of the biocompatibility and intake transport mechanism of nanosystems in vitro and in vivo,it is expected to provide reference for the application of nanosystems in the treatment of inner ear diseases.Previous studies have synthesized composite nanoparticles(NPs)based on the interaction of CMCS/CC-CS NPs and mPEG-PLGA NPs,which have the advantages of multiple response and dual drug-loaded combination therapy,However,studies on the application of such NPs and this type of compound NPs in inner ear delivery are very scarce.The CMCS/CC-CS@mPEG-PLGA NPs loaded with nile red and coumarin-6 were used as the delivery system to study the biocompatibility and uptake in vitro and in vivo.In vitro cytotoxicity and uptake results indicated that CMCS/CC-CS@mPEG-PLGA NPs revealed slight cytotoxicity in the concentration range of less than 20 mg/mL,which could be efficiently taken up by HEI-OC1 cells and distributed around the nucleus,and presented a certain time and concentration dependence.The zebrafish model was used to study the embryotoxicity and uptake of CMCS/CC-CS@mPEG-PLGA NPs.The results showed that CMCS/CC-CS@mPEG-PLGA NPs accelerated the development of zebrafish embryos,and the effect was concentration-dependent.Moreover,CMCS/CC-CS@mPEG-PLGA NPs were effectively ingested by the lateral hair cells of zebrafish.The biocompatibility and distribution of CMCS/CC-CS@mPEG-PLGA NPs in inner ear tissues were studied by using guinea pigs as experimental animal models.Safety of CMCS/CC-CS@mPEG-PLGA NPs was evaluated for inner ear administration through hematoxylin-eosin staining,and both of them showed good biocompatibility.In vivo distribution results demonstrated that CMCS/CC-CS@mPEG-PLGA NPs can be effectively ingested by sensory hair cells in vestibular tissues and cochlear tissue after local administration.In addition,ingestion of vestibular tissue was significantly larger than cochlear tissue.In order to further study the mechanism of inner ear distribution in the nano delivery system,the distribution of CMCS/CC-CS@mPEG-PLGA NPs in the RWM was investigated by using guinea pigs as models.It was found that the green fluorescence of coumarin-6 and the red fluorescence of nile red were widely distributed in the RWM by LSCM,indicating that the NPs could enter the RWM.The perilymph collected after local administration was observed by TEM,and it was revealed that the NPs entered the perilymph in a complete form after local administration.The concentration dependence of the NPs across the RWM was investigated by employing three concentration points of 1.7 mg/mL,5 mg/mL and 15 mg/mL.The results show that the fluorescence intensity of NPs in RWM shows a certain concentration dependence.And the time dependence of the NPs across the RWM was investigated at 15 min,30 min and 60 min.The results revealed that at 0-30 min,the fluorescence intensity of the NPs increased with time in the RWM,but decreased with time at 30-60 min.In order to study the transport mechanism of CMCS/CC-CS@mPEG-PLGA NPs across RWM,the RWM of guinea pigs was observed after intratympanic injection of CMCS/CC-CS@mPEG-PLGA NPs for 30 min by TEM.Compared with the normal control group,a large number of endocytosis and exocytosis vesicles were found in the epithelial cells and the endothelial cells of the RWM in the NPs-treated group,and the tight connection of the epithelial cells of the RWM was widened.The results indicate that CMCS/CC-CS@mPEG-PLGA NPs enter the RWM via intercellular and transcellular pathways.Subsequently,different endocytic inhibitors and intracellular transport inhibitors were used to investigate the specific mechanism of the transcellular pathway.Our data corroborated that the micropinocytosis,caveolin-mediated endocytosis,clathrin-mediated endocytosis were the main way of the NPs into the RWM,and then entered the lysosome,finally,excreted by Golgi body-mediated exocytosis from cells into the perilymph.The RWM acts as a route for substances from the middle ear to the inner ear,but it is also a biomembrane barrier to prevent substances from entering the inner ear.As a cell penetrating peptide,low molecular weight protamine(LMWP)can mediate biological macromolecules and NPs cross biomembrane,providing a new idea for NPs to overcome biomembrane barrier.LMWP was modified on PLGA NPs to investigate the in vitro and in vivo behavior of the delivery system applied to the inner ear.The results of cytotoxicity experiments showed that LMWP-modified PLGA NPs had low cytotoxicity,and only at high concentrations(?2000 ?g/ml),LMWP-modified PLGA NPs showed significant cytotoxicity.The results of cell uptake experiments showed that the uptake of LMWP-modified PLGA NPs in HEI-OC1 cells was significantly higher than that of unmodified PLGA NPs,and the LMWP-modified PLGA NPs were significantly distributed in the nucleus at 30 and 180 min.In vivo animal experiments,after local administration,quantitative analysis of small animal in vivo fluorescence imaging showed that LMWP could promote NPs entry into the cochlea.Then,the distribution of NPs in the RWM was investigated,and the results showed that LMWP could promote NPs entry into the RWM.Meanwhile,it was found that LMWP labeled with rhodamine B was only distributed in the outer epidermis of the RWM,while coumarin-6-labeled PLGA NPs were distributed in the RWM as a whole.CSLM showed that there was no significant difference in the distribution of unmodified PLGA NPs and LMWP-modified PLGA NPs in the cochlear sensory hair cells.In summary,this study systematically studies the biocompatibility and uptake distribution of CMCS/CC-CS@mPEG-PLGA NPs in vitro and in vivo,and clarifies the transport mechanism of the NPs across the RWM.It has important theoretical guiding significance to improve transport efficiency and treatment of inner ear diseases.The application of LMWP modified PLGA NPs to inner ear can effectively increase the uptake and distribution in vitro and in vivo,provide reference for optimizing the design of inner ear delivery system.At the same time,it also provides an important research basis for obtaining better efficiency across the biomembrane barrier.